Store-operated Ca2+ entry (SOCE) underlies numerous cellular processes throughout the body and initiates signaling cascades in T lymphocytes that cause changes in motility, secretion of cytolytic granules, cytokine release, and cell proliferation. The channels that underlie SOCE have been identified recently through RNA interference (RNAi) screening as a conserved family of four transmembrane-spanning proteins named Orai that are activated by STIM proteins in the ER membrane. Isoforms of these proteins are expressed throughout the body in a tissue-specific manner. Important cellular functions of Orai1 have been identified in lymphocytes, microglia, mast cells, blood platelets, sweat and salivary glands, dentition, vascular smooth muscle, endothelial cells, skeletal muscle, microglia, astrocytes, and neurons. In the immune system, STIM1 and Orai1 mediate antigen-induced Ca2+ signaling, motility inhibition at the site of antigen presentation, secretion of cytolytic granules by CD8+ T cells and NK cells, and gene expression responses that lead to cytokine release and cell proliferation. STIM and Orai proteins are being developed as targets for treatment of autoimmune diseases and prevention of transplant rejection. Our overall goal is to understand how Orai channels function at the molecular and cellular level. Orai channels in the plasma membrane are unrelated to other known ion channels and have unusual characteristics that distinguish them, including a very high degree of selectivity for Ca2+, low single-channel conductance, and activation by binding of a small cytosolic domain of the STIM protein. Moreover, the human Orai1 and Orai3 proteins differ in their activation requirements and tissue distribution. In this project, we have three goals. We seek to understand at molecular, subcellular, and cellular levels: 1) how Orai1 is activated by STIM1 at the single-channel level; 2) how Orai1 signals locally in puncta to generate localized Ca2+ signals; 3) how local Ca2+ signals modulate T cell motility, turning behavior and stopping during immune surveillance. To accomplish these Aims, we have developed and continue to develop new tools for monitoring local Ca2+ signals that will be broadly applicable. Our studies will include electrophysiological analysis, optical imaging of Ca2+ flux through Orai1 channels, and two-photon imaging of in situ cellular motility. Overall, our project will provide fundamental insights into the Orai1 proteins that are currently being targeted for treatment of autoimmune disorders, chronic inflammatory conditions, and neurodegerative diseases such as Alzheimer?s.